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Behrooz Azizi, Alireza Ziaei, Thomas Fuchsluger, Thore Schmedt, Yuming Chen, Ula V. Jurkunas; p53-Regulated Increase in Oxidative-Stress–Induced Apoptosis in Fuchs Endothelial Corneal Dystrophy: A Native Tissue Model. Invest. Ophthalmol. Vis. Sci. 2011;52(13):9291-9297. doi: 10.1167/iovs.11-8312.
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This study compared susceptibility of Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs) to oxidative stress, and studied the mechanism of oxidative-stress–induced apoptosis in FECD-affected endothelium.
For in vitro studies, immortalized normal and FECD human corneal endothelial cell lines (HCECi and FECDi, respectively) were exposed to tert-butyl hydroperoxide (tBHP). Apoptotic cell populations were distinguished using flow cytometry. Reactive oxygen species production was measured by a horseradish peroxidase assay. For ex vivo studies, CECs were exposed to tBHP. Oxidative DNA damage and apoptosis were assessed by anti–8-hydroxydeoxyguanosine antibody and TUNEL assay, respectively. p53 and phospho-p53 levels were assessed by Western blot and immunohistochemistry.
Flow cytometry revealed a higher rate of apoptosis in FECDi than that in HCECi after exposure to 0.5 mM (P = 0.010) and 1.0 mM tBHP (P = 0.041). Further analysis showed increased production of H2O2 by FECDi than that by HCECi. Oxidative DNA damage increased in both normal and FECD CECs after exposure to 0.5 mM tBHP (P = 0.031 and 0.022, respectively), leading to a 21% increase in TUNEL-positive CECs in FECD (P = 0.015) but no change in normal. Baseline p53 expression was twofold higher in FECD than that in normal endothelium (P = 0.002). Immunofluorescence revealed an increase in p53 and phospho-p53 levels in FECD compared with that in normal endothelium.
FECD CECs are more susceptible to oxidative DNA damage and oxidative-stress–induced apoptosis than normal. Increased activation of p53 in FECD suggests that it mediates cell death in susceptible CECs. The authors conclude that p53 plays a critical role in complex mechanisms regulating oxidative-stress–induced apoptosis in FECD.
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